Multiple bispecific antibody formats have been proposed and are currently under development. One such format is based upon a standard fully human IgG antibody having an improved pharmacokinetic profile and minimal immunogenicity (see U.S. Pat. No. 8,586,713, which is incorporated herein in its entirety). A single common light chain and two distinct heavy chains combine to form the bispecific. One of the heavy chains contains a substituted Fc sequence (hereinafter “Fc*”) that reduces or eliminates binding of the Fc* to Protein A. For example, one such Fc* sequence contains H435R/Y436F (by EU numbering system; H95R/Y96F by IMGT exon numbering system) substitutions in the CH3 domain. As a result of co-expression of the two heavy chains and the common light chain, three products are created: two of which are homodimeric for the heavy chains and one of which is the desired heterodimeric bispecific product. The Fc* sequence allows selective purification of the FcFc* bispecific product on commercially available affinity columns, due to intermediate binding affinity for Protein A compared to the high avidity FcFc heavy chain homodimer, or the weakly binding Fc*Fc* homodimer.
To achieve commercial scale purification of the bispecific heterodimer, good resolution between the FcFc homodimer, the Fc*Fc heterodimer, and the Fc*Fc* homodimer is required. Here, Applicants describe an improved separation process that optimizes resolution of these three molecular forms.